This Program entitled "Anti-viral Immune Responses in Lymph Nodes" seeks to gain a deeper understanding of the induction and regulation of immunological events that are elicited by viral antigens in lymph nodes (LNs). Many viruses and most conventional vaccines enter the body through the skin and travel via lymphatics to draining LNs. These organs are believed to have a critical role in the adaptive immune response to peripheral infections;therefore, understanding the cellular and molecular interactions that occur within LNs will provide insights that may be useful for improved vaccine development. Lymph-borne foreign matter entering LNs is captured and processed by antigen-presenting cells (APCs), which then present this material to B and T cells to elicit effector responses and long-lived immunological memory. The rules that determine how lymph-derived antigenic material is handled in LNs, especially in the context of ongoing infections, and what cells and molecules must interact to elicit a protective immune response (or fail to do so) are poorly understood. Why do some viruses (e.g. VSV in mice) induce a potent, multi-pronged protective immune response that eliminates the pathogen, while others (e.g. influenza) generate only transient protective immunity, and a few (e.g. HIV in humans) establish a chronic presence by continuously subverting and eventually exhausting the host's anti-viral defenses? The mechanisms behind these different outcomes are likely multi-factorial and depend upon differences in the way individual viruses interact with their hosts. To explore the dynamics of these interactions in living animals, all component projects of this Program will employ multi-photon intravital microscopy (MP-IVM) in intact LNs that will be offered in the Intravital Microscopy Core for time-and space-resolved visualization of the innate and adaptive immune response to lymph-borne virions. In Project 1, Dr. von Andrian will explore innate and adaptive immune responses to lymph-borne viral infections using VSV and several other viral pathogens. In Project 2. Drs. Sharpe and Wherry will visualize the effects of the negative costimulatory pathways, PD-1:PD-L1, PD-1:PD-L2 and PDL1 :B7.1 on antiviral immunity to influenza and LCMV. In Project 3. Dr. Carroll will dissect the role of complement and complement receptors in humoral immunity to influenza virus. Finally, in Project 4, Drs. Luster. Mempel and Tager will characterize the cellular dynamics and viral dissemination in HIV-infected lymph nodes of humanized mice. It is hoped that the mechanistic insights gained from these highly interactive and synergistic experiments will lead to new, evidence-based and knowledge-driven development strategies for anti-viral vaccines. PROJECT 1: Innate and Adaptive Immune Responses to Lymph-Borne Infections (von Andrian, U) PROJECT 1 DESCRIPTION (provided by applicant): Viruses and antiviral vaccines that enter the body through the skin or mucous membranes gain access to nearby lymph vessels and are transported to regional draining lymph nodes (LNs). When a virus arrives in a LN it must be prevented from continuing further along the lymph conduits, which would channel the pathogen into the systemic circulation causing viremia with potentially fatal consequences. LNs are believed to retain and neutralize lymph-borne micro-organisms, but the mechanisms are poorly understood. LNs also initiate adaptive immune responses to viral infections and vaccines because they recruit and harbor large numbers of lymphocytes and specialized antigen presenting cells (APCs), which elicit protective effector responses, especially neutralizing antibodies. Our understanding of how lymph-borne viruses are presented to B cells is still very sketchy. Here, we prepose to use multi-photon intravital microscopy (MP-IVM) for time-and space-resolved visualization of the handling of lymph-borne fluorescent virions in LNs. We hope to obtain mechanistic insights into the cellular and molecular mechanisms by which LNs prevent pathogen dissemination and initiate adaptive humoral immunity. Based on preliminary work, our central hypothesis states that intranodal lymph conduits contain specialized cells that are highly adept at capturing viral particles and at presenting these particles to follicular B cells. This hypothesis will be tested in two specific aims: Aim 1 will explore the cellular mechanisms that determine the fate of lymph-borne viruses in LNs draining a subcutaneous injection site. Preliminary work has identified specialized macrophages (Mphs) in the floor of the subcaspular sinus (SCS) and the medulla of LNs that may function as a cellular 'flypaper'for viral particles and may be responsible for the filter activity of LNs. We will examine the phenotype and function of these virus-capturing Mphs (subaim 1.1) and address their role in the clearance and retention of different lymph-borne viruses (subaim 1.2). We will also investigate the origin and fate of LN-resident Mphs and other innate leukocytes and their relationship to a population of hematopoietic stem and progenitor cells that recirculate continuously between blood, tissues and lymph (subaim 1.3). Aim 2. will analyze where and how follicular B cells are exposed and respond to lymph-borne viruses. We will examine the sequential steps that are elicited by vesicular stomatitis virus (VSV) to induce early T-independent and late T-dependent humoral immunity in LNs. Subaim 2.1 will test the hypothesis that Mphs in the SCS function as early APCs for B cells. Subaim 2.2 will analyze T cell activation and B:T interactions as well as germinal center responses to viral challenge. Finally, subaim 2.3 will explore the possibility that polymeric nanoparticles could be devised to mimic invading viruses and provide a potent and versatile immuno-stimulatory platform for the delivery of future vaccines.